The present invention is related to results obtained from research on the formulation of deoxyribonuclease, otherwise referred to as DNase, a phosphodiesterase that is capable of hydrolyzing polydeoxyribonucleic acid (DNA).
The present invention relates generally to the preparation of liquid solutions of DNase that are protected from thermally induced aggregation of the DNase active principal component. The present invention relates additionally generally to the preparation of liquid solutions of DNase that are maintained stable at pHs of less than neutral.
It relates to these solutions per se and to their methods of preparation and to their use clinically or for preparing further formulations useful clinically in the treatment of disorders susceptible to the biological activity of DNase, as discussed in more detail infra.
DNase is a phosphodiesterase capable of hydrolyzing polydeoxyribonucleic acid. DNase has been purified from various species to various degrees. The complete amino acid sequence for a mammalian DNase was first made available in 1973. See, e.g., Liao, et al., J. Biol. Chem. 248, 1489 (1973).
DNase has a number of known utilities and has been used for therapeutic purposes. Its principal therapeutic use has been to reduce the viscoelasticity of pulmonary secretions in such diseases as pneumonia and cystic fibrosis, thereby aiding in the clearing of respiratory airways. See, e.g., Lourenco, et al., Arch. Intern. Med. 142, 2299 (1982); Shak, et al., Proc. Nat. Acad. Sci. 87, 9188 (1990); and Hubbard, et al., New England Journal of Medicine 326, 812 (1992).
DNA encoding human DNase has been isolated and sequenced and that DNA has been expressed in recombinant mammalian host cells, thereby enabling the production of human DNase in mammalian commercially useful quantities. See, e.g., Shak, et al., Proc. Nat. Acad. Sci. 87, 9188 (1990). Recombinant human DNase (rhDNase) has been found to be useful clinically, especially in purified form such that the DNase is free from proteases and other proteins with which it is ordinarily associated in nature.
The means and methods by which human DNase can be obtained in pharmaceutically effective form is described in the patent applications cited above. Various specific methods for the purification of DNase are known in the art. See, e.g., Khouw, et al., U.S. Pat. No. 4,065,355, issued Dec. 27, 1977; Markey, FEBS Letters 167, 155 (1984); and Nefsky, et al., Euro. Journ. Biochem. 179, 215 (1989).
The present application is predicated on the use of such DNase for formulation. DNase can be employed as such, as a mixture of deamidated and non-deamidated forms, or in isolated deamidated and non-deamidated forms. The preparation and separation of such forms are the subject matter of a patent application cited above.
The present invention is directed to the preparation of liquid solutions of DNase (including all of its biologically active forms as previously noted) that are stable to thermally induced aggregation of DNase.
The present invention is directed to the preparation of stabilized liquid solutions of DNase (including all of its biologically active forms as previously noted). These liquid solutions containing DNase in substantially non-deamidated form are maintained at pHs of less than neutral in stable form such that precipitation of material does not occur to any substantial extent, and therefore, the solutions are in a clear form suitable for pharmaceutical administration. Such less than neutral pH levels result in a reduction of the rate of deamidation of the DNase principle during storage. At storage at elevated temperatures (upwards of 37xc2x0 C.), such lower pH solutions result in precipitation products. The present invention, in an aspect, stabilizes such solutions from such precipitation.
The present invention is predicated upon the finding of an exceptional characteristic found attributable to a particular component which in liquid solution together with DNase as biologically active principle, protects said DNase from thermally induced aggregation. This particular component in liquid solution together with DNase as biologically active principle protects and stabilizes the solution from precipitation effects resulting in clear solutions which are suitable for pharmaceutical administration.
The pharmaceutical specifications permitting the storage with suitable shelf life of DNase requires the retention of at least 80% biological potency at from 2 to 8xc2x0 C. over a sustained period of time. The recommended pH of such solutions are about neutral, more particularly approximately 6.5. It has been discovered that at this pH range, deamidation occurs at a relatively constant rate resulting in solutions in which the DNase component is increasingly deamidated. The deamidation takes place at the asparagine residue that occurs at position 74 in the amino acid sequence of native mature DNase. Attention is directed to U.S. Ser. No. 07/895,300, filed Jun. 8, 1992. In that application attention is focused on the separation of deamidated and non-deamidated DNase from one another for separate formulation into pharmaceutically administrable forms.
In this aspect of the present invention, it has been found that lowering the pH of such liquid solutions containing DNase substantially reduces the rate constant of deamidation resulting in liquid formulations which are relatively stable as to deamidation and thus the DNase remains in its non-deamidated form which is biologically more potent. However, the reduction of pH results in a by-process of precipitation of materials from the liquid solutions when stored at about 37xc2x0 C., which is unacceptable from a pharmaceutical formulation standpoint.
The present invention in this aspect relates to the stabilization of such less than neutral pH liquid solutions containing DNase from precipitation resulting in solutions that are pharmaceutically administrable.
The introduction and use of calcium ion (Ca+2) protects such less than neutral pH liquid solutions of DNase from precipitation resulting in clear solutions that are suitable for pharmaceutical storage and administration, without the need for refrigeration. Thus, the present invention in this aspect relates to a method of stabilizing liquid solutions containing DNase as active principle, such solutions being at a pH of less than neutral such that deamidation is deterred or inhibited which comprises employing amounts of calcium ion in said solution that protect such solutions from precipitation resulting in liquid formulations of DNase that are suitable for storage and ultimate pharmaceutical administration. These solutions are maintained in a stable form without any or any substantial precipitation that otherwise occurs at pHs of less than neutral. Further, these solutions result in a minimal deamidation of the active component of DNase.
In the alternative aspect of the present invention, in signal distinction from certain other divalent cations, calcium ion (Ca+2) additionally protects DNase from thermally induced aggregation in liquid solution. Thus, the present invention relates to a process of minimizing thermally induced aggregation of DNase in liquid solution comprising DNase as active principle which comprises employing DNase-aggregation-minimizing amounts of calcium ion in said solution.
In a further embodiment of this alternative aspect of this invention, protection of DNase from thermally induced aggregation in liquid solution is obtained by the addition of sugars to said solutions, either in lieu of or additional to the presence of calcium ion.
Thus, the present invention is directed to methods for the preparation of liquid solutions comprising DNase as active principle which comprising using in said solutions an amount of calcium ion and/or sugar that: 1) minimizes DNase aggregation brought about from thermal instability (a DNase-aggregation-minimizing amount) and an amount of calcium ion that 2) stabilizes said solutions that are at a pH of less than neutral from precipitation resulting in said solutions being clear and thus in a form suitable for storage and pharmaceutical administration. In the latter, deamidation of the DNase is inhibited, a consequence of the lowered pH (from neutral).
Stated another way, the present invention is directed to a method for the preparation of a liquid solution comprising DNase as active principle which comprises utilizing in said solution an amount of calcium ion and/or sugar that stabilizes said solutions from both thermally induced DNase aggregation and an amount of calcium ion that stabilizes said solutions from precipitation effects when said solutions are at less than neutral pH. In the latter instance, deamidation of the DNase is inhibited, a result of the lowered pH of the solution.
The present invention is further directed in different aspects to the solutions themselves comprising DNase as active principle and amounts of calcium ion, and to the use of these solutions for the treatment of disorders in which the biological activity of DNase can be exploited. As well, it is directed to methods for the use of such solutions in the preparation of further formulations comprising DNase as active principle such as subjecting said solutions to elevated temperatures, e.g. as in spray-drying techniques to produce pharmaceutically acceptable formulations of DNase in the form of a respirable DNase-containing powder, suspension or solution that is therapeutically effective when administered into the lung of an individual. Further, such solutions that are at less than neutral pH, in order to inhibit deamidation of the DNase, are rendered stable to (lower pH) induced precipitation when stored at temperatures at about ambient temperature or above.
The present invention is further directed to all associated embodiments thereof relating to the preparation and use of liquid solutions of DNase, which is essentially in monomeric form and/or inhibited in deamidation, and to stabilize against precipitation at pHs of less than neutral, employing the calcium cation to minimize thermal aggregation of the DNase.
The liquid solutions hereof comprise as essential components DNase as the biologically active principle and a source of calcium cation. In such solutions the DNase is in substantially monomeric form, and where such solutions are at a pH of less than neutral, deamidation of the DNase is inhibited, and in those instances, the solutions are stabilized against precipitation when stored at ambient or elevated temperatures.
The calcium ion source can be virtually any calcium salt supplied directly or formed in situ from a suitable calcium source that is pharmaceutically acceptable such as calcium chloride, in its various hydrated and anhydrous forms, calcium oxide and calcium carbonate. The calcium source for the calcium cation active component of the present liquid solutions of DNase is present generally at a concentration of from about 1 mM to about 1 M, and more preferably, from about 10 mM to about 100 mM.
The sugars may include, for example, xcex1-lactose monohydrate, mannitol, trehalose, sucrose, and the like. They are employed in concentrations generally of from about 50 to about 200 mg/ml., although concentrations outside of this range can also be used.
The solutions hereof may contain other components, such as excipients, with the only requirements being that such other components are pharmaceutically acceptable and do not interfere with the effect of calcium cation.
The solutions hereof can be used as such, or they can be used for the preparation of pharmaceutically acceptable formulations comprising DNase that are prepared for example by the method of spray-drying the liquid solutions hereof and collecting the spray-dried product as a dispersible DNase-containing powder that is therapeutically effective when administered into the lung of an individual. The calcium ion protecting effect applies to any situation in which rhDNase solutions are exposed to elevated temperature and/or where such solutions are at a pH of less than neutral.
For spray-drying the latter embodiment, attention is again directed to the co-pending U.S. application Ser. No. 08/206,020 filed Mar. 4, 1994 for the details concerning the spray drying procedure.
Present results would indicate that minimization of thermal-induced aggregation of DNase in solution can be achieved using lower temperatures in the range less than about 60xc2x0 C. and protein concentrations of about less than 1 milligram per milliliter, with a pH of about 6 to 7 and a concentration of excipient of more than about 10 millimoles (at the concentration of DNase given above).
Present results would indicate that minimization of precipitation of DNase solutions at a pH of less than neutral (in which solutions deamidation of the DNase is inhibited) results in solutions that can be pharmaceutically administered where such solutions are stable over long periods of time, remaining clear for ultimate such pharmaceutical administration.
The DNase formulations hereof are employed for enzymatic alteration of the viscoelasticity of mucus within the lung. Such formulations are particularly useful for the treatment of patients with pulmonary disease who have abnormal viscous, purulent secretions and conditions such as acute or chronic bronchial pulmonary disease, including infectious pneumonia, bronchitis or tracheobronchitis, bronchiectasis, cystic fibrosis, asthma, tuberculosis and fungal infections and the like. For such therapies, the novel formulations hereof are instilled generally by methods familiar to those skilled in the art into the bronchi of the individual being treated. The formulations hereof are particularly suited for the assured introduction into the lung of DNase such that a therapeutically effective amount of DNase is delivered to the individual by direct action in the lung.